APPLIED

AND

ENVIRONMENTAL MICROBIOLOGY, Dec. 1992,

p.

Vol. 58, No. 12

3792-3798

0099-2240/92/123792-07$02.00/0 Copyright © 1992, American Society for Microbiology

Cloning and Expression of a Conjugated Bile Acid Hydrolase Gene from Lactobacillus plantarum by Using a Direct Plate Assay H. CHRISTIAENS,l R. J. LEER,2 P. H. POUWELS,2 AND W. VERSTRAETEl* Laboratory of Microbial Ecology, Faculty of Agricultural Sciences, University of Ghent, Coupure Links 653, B-9000 Ghent, Belgium, 1 and Medical Biological Laboratory, TNO, 2280 AA Rijswijk, The Netherlands2 Received 8 May 1992/Accepted 21 September 1992

The conjugated bile acid hydrolase gene from the silage isolate LactobaciUlus plantarum 80 was cloned and expressed in Escherichia coli MC1061. For the screening of this hydrolase gene within the gene bank, a direct plate assay developed by Dashkevicz and Feighner (M. P. Dashkevicz and S. D. Feighner, Appl. Environ. Microbiol. 53:331-336, 1989) was adapted to the growth requirements of E. coli. Because of hydrolysis and medium acidification, hydrolase-active colonies were surrounded with big halos of precipitated, free bile acids. This phenomenon was also obtained when the gene was cloned into a multicopy shuttle vector and subsequently reintroduced into the parental Lactobacillus strain. The cbh gene and surrounding regions were characterized by nucleotide sequence analysis. The deduced amino acid sequence was shown to have 52% similarity with a penicillin V amidase from BaciUlus sphaericus. Preliminary characterization of the gene product showed that it is a cholylglycine hydrolase (EC 3.5.1.24) with only slight activity against taurine conjugates. The optimum pH was between 4.7 and 5.5. Optimum temperature ranged from 30 to 45°C. Southern blot analysis indicated that the cloned gene has similarity with genomic DNA of bile acid hydrolase-active LactobaciUus spp. of intestinal origin.

It is generally accepted that the indigenous microbiota of the intestinal tract intensively interact with host-produced substances (25) such as mucins (11, 16) and bile acids (5, 12). However, the impact on the host physiology as well as the microbial ecological significance of these interactions is not yet fully understood. Among the various microbial transformations of bile acids, bile acid hydrolysis is a common reaction in the intestinal tract of animals. The conjugated bile acid hydrolase (CBH) (12) enzyme catalyzes the hydrolysis of the amide bond that conjugates bile acids to glycine or taurine. It has been demonstrated that this microbial activity is related to growth depression in chickens (4, 5) and the small bowel syndrome in humans (9, 19). The deconjugation activity is expressed by a large number of intestinal genera (12). Interestingly, the genus Lactobacillus, dominant in the proximal tract of pigs, fowl, and rodents and generally regarded as protective to the host, is also endowed with CBH activity (7, 15, 32). Moreover, from their studies with gnotobiotic mice, Tannock et al. (28) concluded that lactobacilli contribute 74 and 86% of total bile acid hydrolase activity in the cecum and ileum, respectively, of mice. Several CBH enzymes have been purified and characterized from the following genera: Bacteroides (14), Clostridium (8), and (recently) Lactobacillus (15). Yet, the ecological significance of bile acid hydrolysis, particularly which advantage this activity offers the CBH-active bacterium, is not understood. In order to unravel these basic questions concerning gastrointestinal microbial ecology, we intend to study the molecular genetics of CBH. In this report, we describe the first isolation of a conjugated bile acid hydrolase gene from a Lactobacillus plantarum strain, by the use of a direct CBH plate assay adapted *

Corresponding author. 3792

from the method of Dashkevicz and Feighner (3). The nucleotide sequence of the cbh gene and the deduced amino acid sequence are described. Furthermore, the gene was cloned in an Escherichia coli-Lactobacillus shuttle vector and reintroduced into the parental Lactobacillus strain. By this way, overproduction of the CBH enzyme was obtained. MATERIALS AND METHODS

Materials. Sodium salts of taurocholic, taurodeoxycholic (TDCA), taurochenodeoxycholic, glycocholic, glycodeoxycholic (GDCA), and glycochenodeoxycholic acids were obtained from Sigma. Restriction endonucleases, T4 DNA ligase, and molecular mass markers for DNA gel electrophoresis were purchased from Boehringer Mannheim. Bacterial strains, plasmids, and growth conditions. Table 1 lists the bacterial strains and plasmids used in this study. Lactobacillus strains were cultured anaerobically at 37°C in MRS broth (Difco) containing either 2 mM GDCA or TDCA. E. coli MC1061 was cultured aerobically in Luria broth. Transformants of MC1061 with pGI4010 (13) were cultivated in Luria broth supplemented with ampicillin (100 ,ug/ml). CBH assays. By using thin-layer chromatography, Lactobacillus strains were tested for bile acid hydrolase activity. Samples (5 ml) of cultures incubated for 24 h were acidified by addition of 0.5 ml of HCl (6 N) and subsequently extracted with 5 ml of diethyl ether. Extracts and appropriate standards were spotted onto silica gel plates. The chromatograms were developed as described by Gilliland and Speck (7). Bile acid hydrolase activity was determined quantitatively by two different methods. In cell-free extracts, bile acid hydrolysis was determined by measuring the amount of glycine or taurine released from the conjugated bile acid. The reaction mixture (5 ml) contained 2 mM conjugated bile acid, 50 mM sodium acetate buffer (pH 5.0), and an aliquot

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TABLE 1. Strains and plasmids used in this study Isolation source or description

Strain or plasmid

L. acidophilus ATCC 4356 L. brevis ATCC 14869 L. crispatus LD5 L. fermentum LC9 L. gasseni ATCC 33323 L. plantarum 80 L. plantarum L877

Reference

or sourcea

ATCC ATCC 32 32 ATCC 26 This work

Human, pharynx Human, feces Chicken, duodenumn Chicken, crop Human, feces Grass silage Pig, feces

2

E. coli MC1061

pHx pGI4010

Gene bank of L. plantarum 80 DNA in pGI4010 PvuII-EcoRI fragment of pBR322 combined with a polylinker and the ery gene of

pLP3537

Composed of the 2.3-kb plasmid from L. pentosus MD353, the ery gene of pE194, and the plasmid pUC19 E. coi-Lactobacillus shuttle plasmid containing the cbh gene of L. plantarum 80

This work 13

pAMtl

pCBH1

a ATCC, American Type Culture Collection,

21

This work

Rockville, Md.

of the isolated enzyme. Zero-order kinetics were obtained when the reaction mixture was incubated at 37°C for 10 min. By addition of 5 ml of 20% trichloroacetic acid, hydrolase activity was inactivated. Precipitated protein was removed by centrifugation, and the taurine or glycine concentration of the supernatant was determined by means of an adapted ninhydrin method (30) as described by Kawamoto et al. (14). Protein concentration was measured by the method of Bradford (1) by using bovine serum albumin as a standard. On whole cells, the activity was determined by measurement of the free bile acid concentration released from the conjugated substrate by using gas-liquid chromatography (17). Overnight cultures of L. plantarum 80 were centrifuged at 6,000 x g for 15 min at 4°C. Cells were washed twice with 100 mM citrate-sodium phosphate buffer (pH 6.2) and resuspended in the same solution to the appropriate cell concentration. The reaction mixture (10 ml) contained 2.5 mM conjugated bile acid, 100 mM citrate-sodium phosphate buffer (pH 6.2), and an aliquot of resting cells. Samples (3 ml) were taken after 1, 5, and 10 min of incubation at 37°C and added to 0.1 ml of 6 N HCl to stop the reaction. Free bile acids were selectively extracted from the acidified reaction mixtures with diethyl ether and, after methylation, analyzed by gas-liquid chromatography. A Delsi Di 200 gas-liquid chromatograph equipped with a flame ionization detector was used. The column was a coiled glass tube (1 m by 2 mm) packed with 3% SP-2250 on 100/120-mesh Supelcoport (Supelco). Carrier gas was N2 at a flow rate of 30 ml/min. The column temperature was 300°C. As an internal standard, lithocholic acid was used. Genetic methods. Restriction enzyme digestion, ligation, and agarose gel electrophoresis of DNA were performed as described by Sambrook et al. (23). Total cellular DNA from Lactobacillus spp. was prepared as described previously (26). A L. plantarum 80 gene bank was made in pGI4010 as described previously (26). Total cellular DNA was partially digested with Sau3AI and fractionated over a sucrose gradient. Fragments of 5 to 8 kbp were ligated to the dephosphorylated BamHI-cleaved pGI4010 vector (13). The gene bank was then transformed into E. coli MC1061. Gel electrophoresis of plasmids demonstrated that most of the Apr E. coli transformants had a L. plantarum DNA insert. Probes used in Southern blot hybridization of genomic DNA were labeled with digoxigenin by using a commercially

available kit (DIG DNA labeling and detection kit, Boehringer Mannheim). Hybridization and probe detection were performed as outlined in the instructions of the supplier, with the exception of low-stringency hybridization, which was performed at 55 instead of 68°C. L. plantarum 80 was transformed by electroporation as described by Josson et al. (13). Transformants were plated on MRS agar medium supplemented with erythromycin (10 ig/ml). The nucleotide sequence of both DNA strands of a 2.1-kb region containing the L. plantarum 80 cbh gene was determined by the chain termination method (24). Restriction fragments of pH3 were cloned in the appropriate M13 vectors (mpl8 or mpl9), and the resulting phages were used for sequencing with M13 universal primer (Boehringer) or specifically synthesized primers. CBH plate assay. For the screening of the deconjugation gene within the strain 80 gene bank, a differential medium for CBH-active E. coli clones was developed, based on the principle of Dashkevicz and Feighner (3). The medium contained (in grams per liter) the following: agar, 15; tryptone, 10; yeast extract, 5; NaCl, 5; CaCl2. 2H20, 0.35; glucose, 10; TDCA, 5; and ampicillin, 0.1. The pH was adjusted to 6.5. CBH activity of the L. plantarum transformants was demonstrated by toothpicking the bacteria onto solid MRS medium supplemented with erythromycin (10 ,tg/ml) and TDCA (5 mg/ml) (3). Enzyme isolation. Hydrolase-positive E. coli clones were cultivated in Luria broth supplemented with ampicillin (100 ,g/ml). Cells were centrifuged and washed twice with physiological solution (NaCl, 8.5 g/liter). In the first step, cells were incubated on ice for 30 min in the following hypertonic solution (millimolar): sucrose, 600; EDTA, 1; Tris, 100 (pH 7.0). After incubation, cells were again harvested by centrifugation, suspended in distilled water, and incubated for another 30 min on ice. After centrifugation, the osmotic fluid was tested for CBH activity. RESULTS Screening and characterization of CBH-active lactobacilli. By using thin-layer chromatography, the Lactobacillus spp. listed in Table 1 were screened for the ability to hydrolyze

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CHRISTIAENS ET AL. TABLE 2. CBH activity of various Lactobacillus strains CBH activity against:

Strain

ATCC 4356 ATCC 14869 LD5 LC9 ATCC 33323 80 L877

TDCA

GDCA

+ -

+ +

-

-

+

+

-

-

+

+ +

-

bile acids (Table 2). Among others, L. plantarum 80 was found to hydrolyze both the glycine and the taurine conjugate. Lactobacillus crispatus LD5 and Lactobacillus gasseri ATCC 33323 showed no CBH activity. Deoxycholic acid (DCA) was the sole product formed by all hydrolytic strains. CBH activity of cells sampled from a stationary-phase culture of strain 80 was determined by measuring the amount of free bile acid released from the conjugated substrate over time. Activity of cells on TDCA and GDCA was estimated to be, respectively, 0.05 and 1.30 ,umol of DCA released per mg (dry weight) of cells per min. Screening for a cbh gene in E. coli. In order to elucidate the genetic basis of deconjugation, a genomic library of L. plantarum 80 was made in pGI4010 and used to transform into E. coli MC1061. Ampicillin-resistant transformants were tested for the presence of a cbh gene. E. coli clones were grown individually on a solid medium with high-glucose and taurineconjugated bile acid (TDCA) concentrations. It was expected that because of medium acidification and deconjugation of TDCA, hydrolase-active colonies would produce copious amounts of precipitated DCA. Incubation overnight resulted in the detection of six positive colonies surrounded with big halos of a white precipitate (Fig. 1). Genetic characterization of the different CBH-active E. coli clones. The six clones were further characterized by restriction enzyme analysis. Only three of six clones harbored a different, but overlapping, insert on pGI4010. Figure 2 compares the restriction maps of plasmids pHi, pH3, and pH6, carrying, respectively, a 7.4-, 8.0-, and 6.0-kb heterologous DNA insert. To localize the gene more precisely, pHl was further digested with HindlIl. The total digest was ligated at random and, after ligation, used to transform E. coli. By using the above plate assay, transformants were again tested for conjugated bile acid hydrolysis. The screening resulted in the detection of two positive subclones, nominated pHlO and pHll, that carried a 2.4-kb HindIII fragment (Fig. 2) in, respectively, the original and opposite orientation. Compared with the original activity of pHi, the halo production by both clones was, however, much less pronounced (results not shown). From the genomic maps of pH6 and pH10, it can be derived that the cbh gene lies within the left HindIII fragment of pH6. Nucleotide sequence analysis. The nucleotide sequence of the region of plasmid pH3 (Fig. 2) expressing CBH activity in E. coli was determined (Fig. 3). The fragment contains a single open reading frame of 972 nucleotides bounded by a methionine start codon ATG and a translation termination codon TAA. The assigned initiation codon ATG is preceded by a typical Shine-Dalgarno sequence, 5'-AGGAGG-3', at a distance of 10 bp from the start codon. Potential Cr70 promoter sequence elements can be indicated at a distance of 120 (-10 element) and 144 (-35 element) nucleotides up-

FIG. 1. Detection of a CBH-active E. coli MC1061 clone, harboring a hybrid plasmid containing the hydrolase enzyme gene. The CBH-active colony produces copious amounts of DCA.

stream of the start codon (Fig. 3). Downstream of the structural gene, at a distance of 34 nucleotides, a palindromic sequence (AG = -18.9 kcal/mol) followed by a stretch of T residues is found, suggesting that this structure may function as a p-independent transcription termination signal (22, 29). The encoded polypeptide (324 amino acids) has a calculated mass of 37,078 Da. The deduced amino acid sequence of the N terminus of the protein does not resemble

pH 1

11 I --I 161

1

I I

l l

la

A

l

dFh

pH3 A

a

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x

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pH 6

ii__ 1

13

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cbh

pH 10

3I_

co

III_ I

I I

1 kb

a ._ 1

.-l

..

cbh

pH 11

VI'

el

II

cbh

FIG. 2. Restriction maps of different hybrid plasmids isolated from CBH-active E. coli clones (pH1, pH3, and pH6) and derived subclones (pH10 and pHll). The thick lines represent the cloned chromosomal DNA fragments from L. plantarum 80. The thin lines represent vector DNA (pGI4010). Symbols: -*, cbh gene; ........ region whose nucleotide sequence was determined.

BILE ACID HYDROLASE GENE CLONING FROM L. PLANTARUM

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1 1

....

44 50

45 LDHHYAIIGITADVESYPLYYDAMNEKGLCIAGLNFAGYADY KKYDADK 93

70

GCAGCAACTATTTGACCATT

ATCGATGTACGTTGATGAACCCCGAGTTTTAGACGCTGCCATTA

MCTAITYQSYNNYFGRNFDYEISYNEMVTITPRKYPLVFRKVEN.. : 1 1 1:I:1:::11 111 1: MLGCSSLSIRTTDDKSLFARTMDFTMEPDSKVIIVPRNYGIRLLEKENVV :::11:1:

1:11:11111

1:1:1 1

51 INNSYAFVGMGSTDITSPVLYDGVNEKGLMGAMLYYATFATYADEPKKGT 100

140

GAAAAGCAGGTTACTTTCACGAAAGTGCACCATTTTATTACGAGGccMCCCTATGGTGTTAGTMG

94 VNITPFELIPWLLGQFSSVREVKKNIQKLNLVNINFSEQLPLSPLHW LV 142 :1

210

:

:11

111:

1:1

101 TGINPVYVISQVLGNCVTVDDVIEKLTSYTLLNEANIILGFAPPLHYTFT 150

GGCAACGCTTTGCAAGTACTCTGTAAMGCCGAGTTTGACTGCTGCCAACGTGATGGCATTGGTGMGC

143 ADKQESIVIESVKEGLKIYDNPVGVLTNNPNFDYQLFNLNNYRALSNSTP 192

280

TAATTGATTTGAGCATGATTAAGTTTCCAGGACATGGAGTAGCGATGGGCACGCTATTCCTGAGTGA

151 DASGESIVIEPDKTGITIHRKTIGVMTNSPGYEWHQTNLRAYIGVTPNPP 200

350

193 QNSFSEKVDLDSYSRGMGGLGLPGDLSSMSRFVRAAFTKLNSLPMQTESG 242

GCAAGTGGCTGATGAATAACTGTCGATAATGAACATGATGGGGTTGCAAGGCTATTGAGCCTCACG

1:::1 ::I 111111:

1:

11:1 1:1

420

201 QDIMMGDLDLTPFGQGAGGLGLPGDFTPSARFLRVAYWKKYTEKAKNETE 250

AGGTAAGGATAAGCAGGTATMAAGCATTGTTATATCGGTTATAAGGTTACMTTGATTATTGGCTCTT -10

243 SVSQFFHILGSVEQQKGLCEVTDGKYEYTIYSSCCDMDKGVYYYRTYDNS 292

-35

490

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560

II:

:11 :1111

1

11

1111

293 QINSVNLNHEHLDTTELISYPLRSEAQYYAVN ...... 325 1:1: :11 301 RISAVSLMAENLNSQDLITFEWDRKQDIKQLNQVNVMS 338

TAATATTATTTCGAGGAGGATTACTAGTTATGTGTACTGCCATAACTTATCAATCTTATTATMTAC A I T Y Q S Y N N Y T

C

:1111:11:

251 GVTNLFHILSSVNIPKGVVLTNEGKTDYTIYTSAMCAQSKNYYFKLYDNS 300

AAMTCTGGATAGATTAATTA=AGAGATGATTTTATGAMAAGCTTATTACTTATCAATC M

3795

630

TTCGGTAGAAATTTCGATTATGAAATTTCATACAATGAAATGGTTACGATTACGCCTAGAAATATCCAC E

D Y

F

N

R

F G

S

I

Y N

M V

E

T

I

T

P

P

K Y

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FIG. 4. Alignment of the amino acid sequences of L. plantarum CBH amino acid sequence (upper line) and B. sphaericus penicillin V amidase. Identical ( ) and conserved (:) amino acids are indicated.

L

TAGTATTTCGTAGGTGGAGMCTTAGATCACCATTATGCAAATTGGATTACTGCTGATGTAGAAG F R K V

V

L D

N

E

H

A

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770

CTATCCACTTTACTACGATGCGATGAATGAAAAAGGCTTGTGTATTGCGGGATTAAATGCAGGTTAT E K G

N

D A M

L Y Y

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L C

I

A G

L N

F A

G

Y

a signal sequence typical for secretory proteins, suggesting an intracellular location of the CBH enzyme. Amino acid sequence similarity. The deduced amino acid sequence of the CBH from L. plantarum 80 is presented in

840

GCTGATTATMAAMTATGATGcTGATMMGTTMTATcAcAccAmGMTTMTTccTTGGTTATTGG Y

D

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GACAATTTTCAAGTGTTAGAGAAGTGAAAAAGAACATACAAAAACTACTTGGTTMTATTATTTTAG F

Q

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V

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N

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Q K L N L V N

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TGAACAATTACCATTATCACCGCTACATTGGTTGGTTGCTGATAAACAGGAATCGATAGTTATTGAAAGT' E S I V I E S E Q

L H

P

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L

P

L

L V

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1050

GTTAAAGAAGGACTAAAAATTTACGACAATCCAGTAGGTGTGTTAACMACATCCTATTTTGACTACC E

K

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L T N

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1120

AATTATTTAATTTGAACAACTATCGTGCCTTATCAAATAGCACACCTCAAAATAGTTTTTCGGAAAAAGT L

N

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Y

N

S

L

S

T

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P

F

S

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1190

GGATTTAGATAGTTATAGTAGAGGAATGGGCGGACTAGGATTACCTGGAGACTTGTCCTCAATGTCTAGA D

L D

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D

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P

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M

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1260

TTTGTCAGAGCCGCTTTTACTAAATTAAACTCGTTGCCGATGCAGACAGAGAGTGGCAGTGTTAGTCAGT K

F T

F V R A A

E

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L

S

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S

S

G

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TTTTCCATATACTAGGGTCTGTAGAACAACAAAAAGGGCTATGTGAAGTTACTGACGGAAAGTACGAATA L G

I

F H

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L

T

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E

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1400

. * * *

TACAATCTATTCTTCTTGTTGTGATATGGACAAGGGAGTTTATTACTATAGAACTTATGACAATAGTCA T

I

S

Y

S

C

D

C

M

D

K

G

V

Y

Y Y

T

R

Y

D

N

Q

S

ATTAACAGTGTCAATTTAAACCATGAGCACTTGGATACGACTGAATTAATTTCTTATCCATTACGATCAG I

N

S

V

L N

N

H

E

H

L

D

T

T

E

L

I

S

Y

P

L

R

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~~~~~~~~~1540 * * AAGCACAATACTATGCAGTTAACTAAAAGCCACTACTGTAATAGTTAAAATTGTT AAAGAGGCAM A Q Y

Y

A

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N*

1610

GTTTGTTATCAGTI,fG----TC--TTAGTATCACTGTTTTCAGAGTAAGTTCCAATGGCTGGCATTGC 1680

TGTTAATATGACACTAGATGATATAAGTTATCATGTTAAGCTCGATGGTAAGTTGGGTTTAATTGATAGA 1748

AAACATAATAGTTATGAGATGCTTGAAATACCTACTAAAAATACATTTACCAAATMATATGATC

FIG. 3. Nucleotide sequence and deduced amino acid sequence of the L. plantanum cbh gene. Putative promoter elements are underlined, and a possible ribosome binding site is indicated with an asterisk. Inverted repeats capable of forming a stem-loop structure are marked by horizontal arrows.

Fig. 3. Comparison of the amino acid sequence with that of other proteins revealed that the L. plantarum CBH enzyme shares extensive similarity with penicillin V amidase of Bacillus sphaenicus (20) (Fig. 4). The percentage of similarity is 33% for identical amino acids and 52% when conservative substitutions are taken into account. No similarity between the deduced amino acid sequence of CBH of L. plantarum and the terminal amino acid sequences of purified CBH enzymes of Bacteroides vulgatus (14) and Clostridium perfringens (8) is found. Overproduction of CBH activity in L. plantarum. An E. coli-Lactobacillus shuttle vector containing the cbh gene was made by ligation of the PstI-SacI fragment of pHl to pLP3537, cleaved with PstI and SacI. This construct was called pCBH1 (Fig. 5). The shuttle plasmid pCBH1 was introduced in L. plantarum 80 by electroporation. CBH activity was clearly observed on MRS agarose medium containing TDCA (0.5% [wt/vol]) (Fig. 6). Whereas the CBH activity of L. plantarum (transformed with pLP3537) encoded by the single chromosomal gene was not detectable, copious amounts of free DCA precipitated around the transformants containing pCBH1. As measured by gas-liquid chromatography, the CBH activity of whole cells of L. plantarum 80 carrying pCBH1 was found to be five times higher than the activity of cells carrying pLP3537. Screening for other cbh genes. Subsequently, the EcoRIlinearized pHlO plasmid was used as a probe in Southern blot analysis. Electrophoretically separated restriction endonuclease digests of genomic DNA isolated from different Lactobacillus strains were hybridized with the digoxigeninlabeled probe. The hybridization pattern of the genomic DNA of the wild-type strain L. plantarum 80 is shown in Fig. 7A. Low-stringency hybridization of DNA from different Lactobacillus spp. yielded the pattern shown in Fig. 7B. All CBH-active strains showed a hybridization band, while no reaction occurred with L. crispatus LD5 and L. gasseri

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CHRISTIAENS ET AL.

'A

Hindu

FIG. 5. Multicopy Lactobacillus-E. coli shuttle vector pCBH1. , vector DNA _, L. plantarum DNA of pHl; -, cbh gene; containing the gram-negative replication region (ori) and marker gene (amp) and the gram-positive replication sequence (p353-2) and marker gene (ery).

ATCC 33323. In control experiments with the pGI4010 vector as a probe, no hybridization was found. Preliminary characterization of the gene product. The CBH enzyme produced by the E. coli clones was released by osmotic treatment of the cells and measured quantitatively by the ninhydrin method. No significant difference could be demonstrated between the CBH activity of the clones harboring pHi, pH3, and pH6 (data not shown). The crude osmotic fluid of clone pHl was chosen to study the kinetic properties of the cbh gene product. Table 3 summarizes the substrate specificity of the isolated enzyme. The enzyme was found to have a 15- to 30-fold-higher activity against glycine conjugates than against taurine conjugates and therefore can be considered as a cholylglycine hydrolase. In pH dependency experiments, the enzyme showed a broad pH optimum over the range of 4.7 to 5.5 (Fig. 8A). Optimum temperature ranged from 30 to 45°C (Fig. 8B). From a Lineweaver-Burk plot, the Km value and maximum rate of metabolism of CBH for GDCA were estimated to be, respectively, 0.22 mM and 1.56 ,umol/mg of total protein extract per min. DISCUSSION Conjugated bile acid hydrolysis is a widespread characteristic among Lactobacillus spp. that are isolated from the alimentary tract (7, 15, 32). This is confirmed by the results listed in Table 2. With the exception of L. crispatus LD5 and L. gasseri ATCC 33323, all strains are able to deconjugate TDCA and/or GDCA. Remarkably, the silage isolate (26), L. plantarum 80, also has CBH activity towards the two types of bile acids. The presence of this enzyme in a Lactobacillus silage isolate may be explained by the physical linkage between the silage ecosystem and the gastointestinal ecosystem (27, 33). To study the molecular and microbial ecology

FIG. 6. Manifestation of CBH activity by L. plantarum transformants on solid medium containing 0.5% (wt/vol) TDCA. The L. plantarum colony in the center is the parental strain containing the pLP3537 plasmid (control). The four colonies surrounded with halos of precipitated DCA are transformants containing the pCBH1 vector.

of conjugated bile acid hydrolysis, L. plantarum 80 is selected because it is known to be easily accessible to genetic engineering (26). To screen for E. coli clones harboring the cbh gene of L.

B

A A1 2 3

1 2 3 4

5 6 7 8

KB. KB.t

:_ 23,1. 6.6t -

9,4.,

6,61...-

4.4

2.3

FIG. 7. Southern hybridization of electrophoretically separated restriction endonuclease digests of genomic DNA preparations. (A) Genomic DNA of L. plantarum 80 digested with EcoRI (lane 2) and HindIII (lane 3); lane 1, Molecular mass marker II (Boehringer Mannheim). (B) Genomic DNA of different Lactobacillus spp. digested with HindIII. Lanes: 1, Molecular mass marker II; 2, strain LD5; 3, strain ATCC 33323; 4, strain LC9; 5, strain ATCC 4356; 6, strain L877; 7, strain ATCC 14869; 8, strain 80.

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(6). Glycine conjugates may partially precipitate without hydrolysis at fermentative pH values. Nevertheless, our results clearly demonstrate that the plate assay, with TDCA as a substrate, can be applied even to detect a cbh gene encoding an enzyme with low specificity against the taurine conjugate. Preliminary characterization of the CBH enzyme 1.71 . ..................... Glycoch4Lenodeoxycholic acid..... (EC 3.5.1.24) indeed shows that it is a cholylglycine hydro0.10 Taurocholic acid .............................. lase with only a slight activity against taurine conjugates 0.10 TDCA.. 0.13 (Table 3). Clearly, the cholyltaurine hydrolase activity of L. Taurochoenodeoxycholic acid .............................. plantarum is too low to detect any hydrolase activity against a 1 U = 1 ~Lmol of glycine or taurine released per min. TDCA in a direct MRS plate assay (Fig. 6, control). However, when the cbh gene was cloned into the plasmid pGI4010 and the shuttle pLP3537 and subsequently transformed in, respectively, E. coli and the parental Lactobacilplantanum 80, a direct plate assay developed by Dashkevicz lus strain, hydrolase activity could be demonstrated on solid and Feiighner (3) was adapted to the growth requirements of medium supplemented with TDCA (Fig. 1 and 6). The high E. coli. The differential medium has a high glucose concencopy number of the plasmid vectors in E. coli probably tration, allowing an acidification below pH 5, and TDCA as compensates for the lack of specificity of the CBH enzyme. a substrrate for conjugated bile acid hydrolysis. Since taurine conjugaLtes exhibit a pKa that is less than 1.0 (10), only the The CBH activity of the E. coli clones harboring pHlO and pH1l was less pronounced because the cloned chromosomal deconjuigated product (pK., 5.0) precipitates at fermentative DNA fragment lacks the cbh gene promoter (Fig. 2 and 3). pH valiaes. The halo formation of precipitated free bile acid The isolated cbh gene and surrounding regions were permits the differentiation between hydrolase-positive and characterized by nucleotide sequence analysis (Fig. 3). The -negati te E. coli clones (Fig. 1). Although L. plantarum cells gene is preceded by sequences that are common to a70 show al 25-fold-higher activity against glycine conjugates promoters in Bacillus spp. and E. coli (18, 22). Therefore, it than ag,ainst taurine conjugates, it was not possible to use a is of no surprise that the L. plantarum cbh gene is expressed glycine substrate because of its relative high pK. value of 3.9 in E. coli on its own regulatory signals. The transcript length predicted from the assigned start and termination points of transcription was confirmed by Northern (RNA) blot analyprotein) (U/mg cbh activi ,ityity sis (results not shown), indicating that CBH is encoded by a 2 ______(U/mg________protein)__________ A monocistronic mRNA. Analysis of the amino acid sequence derived from the L. plantarum cbh gene indicated that this sequence shares ¢ R , extensive similarity with penicillin V amidase from B. sphaericus (Fig. 4). Both enzymes hydrolyze an amide 10 bound and have a pH optimum between 5 and 6. Penicillin V amidase and CBH are produced by a wide range of bacteria, O.' yet the physiological role of the enzymes for the bacteria is still unclear (20, 28, 31). CBH enzymes from C. perfringens I < (8) and B. vulgatus (14) and penicillin V amidase from B. 8 7 6 4 3 5 are reported to occur intracellularly as tetramers, sphaericus pH pH suggesting a similar location and possibly also a similar configuration for CBH of L. plantarum. It is tempting to speculate that CBH in L. plantarum and penicillin V amidase in B. sphaericus act in the same, as yet unknown pathway. Whether these enzymes can hydrolyze their mutual substrates, conjugated bile acids and penicillin V, respectively, iity (U/mg protein) remains to be determined. 2 B Southern blot experiments indicate that the gene is well conserved among the different Lactobacillus spp. that were < s tested (Fig. 7B). This may indicate that the CBH enzyme fulfils an important-but yet unknown-cell function. Fur1 f thermore, our results indicate that the pHlO insert might be \ used as a probe to selectively pick up CBH-active Lactobacillus spp. among intestinal populations. Indeed, no hybrid5 ization was found between the pHlO probe and the DNA from strains lacking CBH activity, i.e., L. crispatus LD5 and L. 0 gasseri ATCC 33323 (Table 2). Finally, hybridization with 0 60 50 30 40 20 Temperoture (00) DNA of the parental strain, L. plantarum 80, indicates the that the CBH enzyme is not plasmid coded. Strain 80 is acetate Sodium known to harbor a plasmid of ±+10.2 kbp and yields, upon FIG. 8. (A) Effects of pH on CBH activity. and 3.5 to 5.5, for the ppH rage was as used range oof 3. .5,and or te buffer ('500500 ued mM) M) with EcoRI, two bands of 4.7 and 5.5 kb (data not digestion w 5.5 k(ta E potassiuim phosphate buffer (500 mM) was used for the pH range of two bdofI4.7eand dshtion shown). Hybridization of the EcoRI-digested DNA with the 6.0 to 8..0. (B) Effects of temperature on CBH activity at pH 5.0. In pHlO probe shows only one band of ±23 kb (Fig. 7A). both ex periments, GDCA was used as the conjugated bile acid In a subsequent study, the cbh gene will be used to substratte. One unit of enzyme activity is defined as 1 ,umol of construct a suicide integration vector. The intent is to use glycine released per min.

TABL_E 3. Substrate specificity of the isolated CBH enzyme Sp act (Ua/mg Substrate of protein) 3.42 Glycoch olic acid. .58 GDCA.. .............................................................. ..............................................................

cbh

-

nth

---- r--

3798

CHRISTIAENS ET AL.

this vector to insertionally inactivate the cbh gene of L. plantarum 80. Chromosomal integration in this strain by single homologous recombination has already been carried out successfully (26). The constructed isogenic strains can then have their conjugated bile acid resistance and overall ecological behavior compared. REFERENCES 1. Bradford, M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. 2. Casadaban, M., and S. N. Cohen. 1980. Analysis of gene control signals by fusion and cloning in Escherichia coli. J. Mol. Biol. 138:179-207. 3. Dashkevicz, M. P., and S. D. Feighner. 1989. Development of a differential medium for bile salt hydrolase-active Lactobacillus spp. Appl. Environ. Microbiol. 55:11-16. 4. Feighner, S. D., and M. P. Dashkevicz. 1987. Subtherapeutic levels of antibiotics in poultry feeds and their effects on weight gain, feed efficiency, and bacterial cholyltaurine hydrolase activity. Appl. Environ. Microbiol. 53:331-336. 5. Feighner, S. D., and M. P. Dashkevicz. 1988. Effect of dietary carbohydrates on bacterial cholyltaurine hydrolase in poultry intestinal homogenates. Appl. Environ. Microbiol. 54:337-342. 6. Fini, A., and A. Roda. 1987. Chemical properties of bile acids. IV. Acidity constants of glycine-conjugated bile acids. J. Lipid Res. 28:755-759. 7. Gilliland, S. E., and M. L. Speck. 1977. Deconjugation of bile acids by intestinal lactobacilli. Appl. Environ. Microbiol. 33:1518. 8. Gopal-Srivastava, R., and P. B. Hylemon. 1988. Purification and characterization of bile salt hydrolase from Clostridium perfringens. J. Lipid Res. 29:1079-1085. 9. Gorbach, S. L., and S. Tabaqchali. 1969. Bacteria, bile and the small bowel. Gut 10:963-972. 10. Hofmnan, A. F., and A. Roda. 1984. Physicochemical properties of bile acids and their relationship to biological properties: an overview of the problem. J. Lipid Res. 25:1477-1489. 11. Hoskins, L. C., and E. T. Boulding. 1981. Mucin degradation in human colon ecosystems. Evidence for the existence and role of bacterial subpopulations producing glycosidases and extracellular enzymes. J. Clin. Invest. 67:163-172. 12. Hylemon, P. B. 1985. Metabolism of bile acids in intestinal microflora, p. 331-343. In H. Danielsson and J. Sjovall (ed.), Sterols and bile acids. Elsevier Science Publishing Inc., New York. 13. Josson, K., T. Scheirlinck, F. Michiels, C. Platteeuw, P. Stanssens, H. Joos, P. Dhaese, M. Zabeau, and J. Mahillon. 1989. Characteristic of a Gram-positive broad host range plasmid isolated from Lactobacillus hilgardii. Plasmid 11:9-20. 14. Kawamoto, K., I. Horibe, and K. Uchida. 1989. Purification and characterization of a new hydrolase for conjugated bile acids, chenodeoxycholyltaurine hydrolase, from Bacteroides vulgatus. J. Biochem. 106:1049-1053. 15. Lundeen, S. G., and D. C. Savage. 1990. Characterization and purification of bile salt hydrolase from Lactobacillus sp. strain 100-100. J. Bacteriol. 172:4171-4177.

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16. Macfarlane, G. T., S. Hay, and G. R. Gibson. 1989. Influence of mucin on glycosidase, protease and arylamidase activities of human gut bacteria grown in a 3-stage continuous culture system. J. Appl. Bacteriol. 66:407-417. 17. Masuda, N. 1981. Deconjugation of bile salts by Bacteroides and Clostridium. Microbiol. Immunol. 25:1-11. 18. Moran, C. P., N. Lang, S. F. J. LeGrice, G. Lee, M. Stephens, A. L. Sonenshein, J. Pero, and R. LosicL 1982. Nucleotide sequences that signal the initiation of transcription and translation in Bacillus subtilis. Mol. Gen. Genet. 186:339-346. 19. Northfield, T. C., B. S. Drassar, and J. T. Wright. 1973. Value of small intestinal bile acid analysis in the diagnosis of the stagnant loop syndrome. Gut 14:341-347. 20. Olsson, A., and M. Uhlen. 1986. Sequencing and heterologous expression of the gene encoding penicillin V amidase from Bacillus sphaericus. Gene 45:175-181. 21. Posno, M., R. J. Leer, N. van Luok, M. J. F. van Giezen, P. T. H. M. Heuvelmans, B. C. Lokman, and P. H. Pouwels. 1991. Incompatibility of Lactobacillus vectors with replicons derived from small cryptic Lactobacillus plasmids and segregational instability of the introduced vectors. Appl. Environ. Microbiol. 57:1822-1828. 22. Rosenberg, M., and D. Court. 1979. Regulatory sequences involved in the promotion and termination of RNA transcription. Annu. Rev. Genet. 13:319-353. 23. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 24. Sanger, F. S., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467. 25. Savage, D. C. 1977. Microbial ecology of the gastro-intestinal tract. Annu. Rev. Microbiol. 31:107-133. 26. Scheirlinck, T., J. Mahillon, H. Joos, P. Dhaese, and F. Michiels. 1989. Integration and expression of a-amylase and endoglucanase genes in the Lactobacillus plantarum chromosome. Appl. Environ. Microbiol. 55:2130-2137. 27. Tannock, G. W. 1990. The microecology of lactobacilli inhabiting the gastrointestinal tract. Adv. Microb. Ecol. 11:147-171. 28. Tannock, G. W., M. P. Dashkevicz, and S. D. Feighner. 1989. Lactobacilli and bile salt hydrolase in the murine intestinal tract. Appl. Environ. Microbiol. 55:1848-1851. 29. Tinoco, I., P. N. Borer, B. Dengler, M. D. Levine, 0. C. Uhlenbeck, D. M. Crothers, and J. Gralla. 1973. Improved estimation of secondary structure in ribonucleic acids. Nature (London) New Biol. 246:40-41. 30. Troll, W., and R. K. Cannan. 1953. A modified photometric ninhydrin method for the analysis of amino and imino acids. J. Biol. Chem. 200:803-811. 31. Vandamme, E. J., and J. P. Voets. 1974. Microbial penicillin acylases. Adv. Appl. Microbiol. 17:311-369. 32. Vandevoorde, L., H. Christiaens, and W. Verstraete. 1992. Prevalence of coaggregation reactions among chicken lactobacilli. J. Appl. Bacteriol. 72:214-219. 33. Van Renterghem, B., F. Huysman, R. Rygole, and W. Verstraete. 1991. Detection and prevalence of Listeria monocytogenes in the agricultural ecosystem. J. Appl. Bacteriol. 71:211217.

Cloning and expression of a conjugated bile acid hydrolase gene from Lactobacillus plantarum by using a direct plate assay.

The conjugated bile acid hydrolase gene from the silage isolate Lactobacillus plantarum 80 was cloned and expressed in Escherichia coli MC1061. For th...
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